Program Director/Principal Investigator (Last, First, Middle): Urayama, Akihiko PROJECT SUMMARY ABSTRACT Effective brain delivery of therapeutic molecules has been a major roadblock in our ability to treat central nervous system (CNS) diseases. We have examined lysosomal enzyme transport across the blood-brain barrier (BBB) which is mediated through the insulin-like growth factor 2/mannose 6-phosphate (IGF2/M6P) receptor. We found that this transport mechanism is down-regulated with development, reducing the ability to transport molecules like lysosomal enzymes across the BBB. Importantly, we have found that this transport mechanism is pharmacologically re-inducible in the adult BBB by utilizing and manipulating the endogenous characteristics of the BBB. This work may lead to a novel treatment strategy for lysosomal storage diseases. In this study, we will investigate strategies to enhance lysosomal enzyme transport across the BBB for the treatment of CNS lysosomal storage diseases. To accomplish this, we will focus our studies on mucopolysaccharidosis (MPS) type VII (Sly?s disease), a well-characterized lysosomal storage disease that results from a deficiency of the enzyme ?-glucuronidase (GUSB), that is not currently treatable. Our preliminary data show that treatment with epinephrine up-regulates this endocytic transport pathway in cultured brain microvessel endothelial cells (BECs), enhancing the cellular uptake of GUSB. In addition, using live cell imaging, we found lysosomal trafficking toward the plasma membrane occurred. We hypothesize that adrenergic re-induction of endosomal IGF2/M6P receptors in brain microvessel endothelial cells (BECs) found at the BBB surface will enhance the therapeutic brain delivery of GUSB. Our goals are to investigate how intracellular desensitization of endocytosis occurs in the adult BBB, and elucidate the mechanisms by which endosomal IGF2/M6P receptor activity can be re-induced at the plasma membrane with epinephrine stimulation. We will focus on Rab11 GTPase-mediated endosomal vesicle trafficking (Aim 1). In Aim 2, we propose to test therapeutically relevant doses of GUSB after pharmacologic induction of transport in MPS VII mice. We will test if the enzyme GUSB administered with epinephrine can ameliorate the abnormal CNS storage of glycosaminoglycans in MPS VII mice. Enzymatic GUSB activity after crossing the BBB in MPSVII mice will be determined by a two-photon imaging approach, by measuring catabolic activity against fluorescent substrate. We will also create primary BEC cultures from MPS VII mice, to assess how lysosomal impairment affects the BBB transport of GUSB using longitudinal live cell imaging. Investigating the effects of pharmacologic manipulations of BBB transport of the enzyme GUSB on CNS lysosome storage in MPS VII mice has direct translational relevance for the management of storage diseases and may have wider significance as a potential mechanism to improve BBB penetrance for other substances. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page